1. Field of the Invention
The invention relates to methods for early diagnosis and staging of cancer which is causatively related to derangements of chromosomal 9p21 in mammals.
2. History of the Related Art
The chromosomal region 9p21 harbors five different genes within about 120 kb: the tumor suppressor genes p15IINK4B (CDKN4B, hereafter xe2x80x9cp15xe2x80x9d) with its alternative spliced form p10, p16INK4A (CDKN2A, a key regulator of the G1-phase of the cell cycle and growth control, hereafter xe2x80x9cp 16xe2x80x9d) and p19ARF (which acts via a p53 dependent pathway to block cell cycle progression), as well as the gene for the metabolic enzyme methylthioadenosine phosphorylase (MTAP).
Homozygous deletion is the most important mechanism of inactivation of all three genes. While somatic and germline mutations have been described for p16, they seem to be very rare in the other two genes. A third mechanism of inactivation is hypermethylation of CpG-islands in the promoter region of both p15 and p16, which results in silencing of transcription. No relationship between hypermethylation and tumor grade has been observed.
Studies of tumor cell lines and gross primary tumors reveal that homozygous deletions of p16 are found in 10-60% of brain tumors, depending on the tumor stage and histology. Homozygous deletions of p16 are also present in melanomas, lung cancers, malignant mesotheliomas, bladder cancers, pancreatic carcinomas, ovarian carcinomas, head and neck cancers, chondrosarcomas, esophageal squamous cell carcinomas, T-cell acute lymphoblastic leukemias, and other primary lymphoid malignancies.
Loss of heterozygosity (LOH) as well as hemi- and homozygous deletions of the 9p21 region encompassing the p161NKA4 gene and MTAP genes have been described in a variety of human tumors, primarily based on analysis of tumor cell lines, including acute lymphoblastic leukemia (ALL), melanoma, ovarian cancer, glioma, head and neck cancer, bladder cancer, chondrosarcoma, small cell and non-small cell lung cancer (NSCLC). In general, it has been widely concluded that the portion of 9p21 where the p16 INK4A gene resides is the point at which derangements in the region of 9p21 between that and the MTAP gene begin, indicating that p16 would be a marker for cancer at early and more advanced stages of tumor development.
The invention provides a method for differentiating among stages of tumor development which is causatively related to derangements of chromosomal region 9p21 in mammals. More particularly, the invention provides a framework for both early diagnosis of such cancers and prognosis based on the stage of tumor development. In the paradigm provided by the invention, derangements of 9p21 in many cancers begin at the MTAP gene locus at the onset of tumor development and progress centromerically toward the p16 gene locus as tumor development advances, as represented in the pictorial below:
According to the method of the invention, a model pattern of deletion of portions of chromosome 9p21 (xe2x80x9cstaging referencexe2x80x9d; see, e.g., FIG. 1) is used as a reference for comparison to 9p21 in samples of cells which are suspected of, or confirmed as, being cancerous. The sample cells are analyzed for the presence of deletions and breakpoints in 9p21. The deletion pattern identified in the sample cells is compared against the staging reference, in which the presence or absence of certain deletions and breakpoints in 9p21 are correlated to the stage of development of cancer in the sample cell population.
The data upon which the staging reference is based are derived from analysis of 9p21 in histological grade, paraffin-embedded samples of human tumors at various stages of tumor development identified according to conventional staging techniques. Unlike the tumor cell lines used in previous analyses of 9p21 derangements, the unique tissue samples utilized as the basis for development of the staging reference include very early stage tumors as well as advanced stage tumors.
Surprisingly, analysis of the tissue samples revealed that, in contrast to widely held perception, the p16INK4a (p16) gene is not the starting point for the 9p21 derangements observed in cancer cell lines and advanced primary tumors. Rather, the derangement starting point in many tumor cells lies just centromeric to exon 8 of the MTAP gene (telomeric to sequence tagged site [STS] 3.21), while in others the first breakpoint is within the MTAP gene, between exons 4 and 5. As tumor development progresses, deletions within 9p21 advance centromerically toward inclusion of all or part of the p16 coding sequence, and may also advance telomerically toward of all or part of the MTAP coding sequence. An additional fragile site lying between p16 and p15INK4b (p15) can lead to deletion of a portion of 9p21 in more advanced tumor cells. Thus, p16 deletion alone is not a reliable marker for either early diagnosis or prognostic staging of cancer, both of which can be accomplished by use of the staging reference of the invention.
The invention also provides methods and reagents for use in early diagnosis of cancer through hybridization to early deletion points in 9p21.